Fig. 2: Development and optimization of RISE selection conditions.
From: In vitro ribosome synthesis and evolution through ribosome display
a iSAT translation of sfGFP under stalling conditions. pRDV-sfGFP plasmids were made with (red) or without (blue) a 3’ HH ribozyme for processing of transcribed mRNA to remove the stop codon. Anti-ssrA oligonucleotide was included at either 0 μM (solid) or 5 μM (dotted). b Sedimentation analysis of iSAT reactions with wild-type or nonfunctional rDNA operon. Peak identities are labeled. c Relative specificity of capture by anti-FLAG magnetic beads for iSAT reactions displaying FLAG-tag. Reactions were incubated from 15 min to 2 h. d Comparison of relative specificity of various bead/tag purification systems for use in RISE. Wash methods were held constant for comparison, and reactions were incubated for 1.5 h, except for reactions with GST, which were incubated for 2 h. For a, c, and d, values represent averages of three independent reactions (n = 3), while shading in a and bars in c and d represent 1 SD.
